All auditory information received by the central nervous system is transferred across the inner hair cell - afferent fiber synapse. Dysfunction of this synapse due to aging, noise or genetic disorders reduces hearing dramatically. This remarkable synapse must transfer intensity and timing information faithfully for long periods of time. Ribbon synapses, like those in hair cells, are typically found in cells that use graded receptor potentials and need to have sustained release. The hair cell ribbon synapse is more robust than most, releasing at higher rates and sustaining release for longer periods of time. In order for synapses to operate in this manner they must have a robust means of recycling and replenishing vesicles to synaptic release sites. We have identified a nonlinear calcium dependent capacitance change that we hypothesize is a reflection of vesicle recruitment and replenishment. Our first major goal (Aim 1) is to determine whether this nonlinear change is in fact synaptically based and we will do that using paired recordings to identify a postsynaptic response, using a new glutamate sensor to determine if glutamate release mimics the capacitance response and by inactivating ribbons optically to see if we can ablate this nonlinear component. We have also identified a calcium induced calcium release process (CICR) associated with the nonlinear capacitance change. We are hypothesizing that vesicle trafficking is calcium dependent and that CICR is used as a means of ensuring continual vesicle supply while calcium at the synapse is tightly regulated. We will use optical, electrophysiological and immunocytochemical tools to investigate this possibility in aim 2. Otoferlin is a unique protein implicated to regulate vesicle fusion, trafficking and reuptake. We are postulating that biochemical modification of this protein via phosphorylation by CaM Kinase II or ubiquitination is responsible for targeting these different functions. We will investigate this hypothesis using electrophysiological, optical, molecular and immunocytochemical technologies.